Development of novel salicylic acid–donepezil–rivastigmine hybrids as multifunctional agents for the treatment of Alzheimer’s disease

Abstract Alzheimer’s disease (AD) is a chronic, progressive brain degenerative disease that is common in the elderly. So far, there is no effective treatment. The multi-target-directed ligands (MTDLs) strategy has been recognised as the most promising approach due to the complexity of the pathogenesis of AD. Herein, novel salicylic acid–donepezil–rivastigmine hybrids were designed and synthesised. The bioactivity results exhibited that 5a was a reversible and selective eqBChE inhibitor (IC50 = 0.53 μM), and the docking provided the possible mechanism. Compound 5a also displayed potential anti-inflammatory effects and significant neuroprotective effect. Moreover, 5a exhibited favourable stabilities in artificial gastrointestinal solution and plasma. Finally, 5a demonstrated potential cognitive improvement in scopolamine-induced cognitive dysfunction. Hence, 5a was a potential multifunctional lead compound against AD.


Synthesis of compounds 3a and 3b
General preparation procedures of compounds 3a and 3b. The starting material 1a or 1b (2,4-dihydroxybenzoic acid 1a; 2,5-dihydroxybenzoic acid 1b) (2 mmol) was dissolved in 10 mL THF. Then, EDCI (3.0 mmol), HOBT (3.0 mmol) and excessive amounts (2.2 mmol) of 4-benzylpiperidine (2) were added into the solution, respectively. The reaction mixture was stirred at room temperature overnight and was monitored by TLC. After the reaction completed, the solvent was evaporated under reduced pressure. The crude residue was dissolved in 30 mL water, washed with CH2Cl2 (2 × 30 mL), the combined organic solvent was washed with saturated NaCl (80 mL) and dried with Na2SO4. The organic solvent was evaporated under reduced pressure to obtain crude residue, which was further purified by silica gel chromatography (petroleum ether/acetone = 30: 1) to obtain the key intermediate compounds 3a and 3b.
BV-2 cells in logarithmic growth stage were digested and centrifuged, and then resuspended in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum. The cells was adjusted to 1.2 × 10 4 cells/well, and then seeded evenly in 96-well cell culture plates. The cells were incubated for 36 h at 37℃ with 5% CO2.
After incubation, the cells were added to different concentrations (10 and 2 μM) of tested compounds, and incubated for 24h at 37℃ with 5% CO2. Then, 10 μL CCK-8 solution was added to each well, and incubated for another 1h at 37℃ with 5% CO2.
BV-2 cells (2 × 10 5 cells/well) were seeded in a 24-well cell culture plates. The cells were divided into blank control group, model group (1μg/mL LPS), LPS + 2 μM compound group, LPS + 1 μM compound group. Each drug group was pretreated with conrresponding concentration of drug solution for 8h. After 8h, the culture medium was aspirated out. Except for the blank control group, which was added with DMEM medium, the other groups were stimulated with 1 μg/mL LPS for 24h. Then cell supernatant was aspirated and centrifuged at 3000 rpm for 3 min. The content of NO in cell culture supernatant was determined by Griess method, and the levels of cytokines IL-6 and IL-1β in cell culture supernatant were determined by IL-6 ELISA kit and IL-1β ELISA kit, respectively.
(4) PC12 Cell Recover. Quickly remove the cells from the -80 °C freezer, shake in a 37 °C water bath pot to dissolve quickly, disinfect the cryopreservation tube and transfer it to the ultra-clean  After aggregation is complete, place at -20 °C for standby and dilute with PBS before use so that the final concentration of its entry into the cells is 25 μmol/L. Seal with plastic wrap and band during culture to prevent loss of moisture evaporation.  overnight and allowed free access to water. Compound 5a at doses of 250, 500 and 1000 mg/kg (n = 6 per group) by intragastric administration. After the administration of the compound 5a, the mice were observed continuously for the first 4 h for any abnormal behavior and mortality changes, intermittently for the next 24 h, and occasionally thereafter for 14 days for the onset of any delayed effects. All animals were sacrificed on the 14th day after drug administration and were macroscopically examined for possible damage to the heart, liver, and kidneys.

In vivo assay
Assay method. The step-down passive avoidance task was employed to investigate the effects of 5a on scopolamine-induced memory impairment. 21,22 Sixty mice were random divide into six groups. They were compound 5a group (9.0 mg/kg, 18.0 mg/kg and 36.0 mg/kg), same volume of water (untreated group), model group (3 mg/kg scopolamine), 10.0 mg/kg rivastigmine. After 30 min, memory impairment was induced by administering scopolamine (3 mg/kg). Then 30 min later, the learning and memory capacity of mouse were measured by the Y-maze test. The maze was made of black-colored acryl and positioned at equal angles. Rats were placed at the end of the arm and allowed to move freely through the maze during 8 min sessions. Arm entry sessions were recorded when the hind paws of the rat were completely placed in the arm. Consecutive entry into three arms in alternative order was defined as successive entries on overlapping triplet sets and the alternation percentage was calculated as the ratio of actual to possible alternations (defined as the total number of arm entries minus 2), multiplied by 100.